Pertussis Culture Research Articles

THE EFFECT OF EARLY ERYTHROMYCIN TREATMENT ON THE INFECTIOUSNESS OF WHOOPING COUGH PATIENTS.

The effect of early erythromycin treatment was studied in 63 whooping cough patients. They were all culture‐positive in the catarrhal stage and/or two weeks or less after the onset of coughing. 28 patients were treated with eryhtromycin for 7–10 days: children with a dose of 40 mg/kg/day and adults with 1g/day. 35 patients received no antibiotics. 152 Bordetella pertussis cultures were obtained from the untreated patients and 70 from the patients treated with eryhtromycin.The erythromycin‐treated patients were culture‐negative on average 1‐2 weeks earlier than the untreated patients. Of the patients treated with erythromycin, 6/28 (21%) were still culture‐positive after treatment. This study shows that erythromycin treatment may reduce the period during which patients are infectious and that the treatment should be started two weeks from the onset of coughing, or preferably earlier.

Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells.

Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity showed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine.

Amphiphile-mediated activation of soluble adenylate cyclase of Bordetella pertussis

The adenylate cyclase activity of Bordetella pertussis culture supernatants is activated 3- to 10-fold by various amphiphiles including many classes of phospholipids and nonionic detergents. Gangliosides are inhibitory. The stimulation affects the V max and not the K m . Neither the nature of the polar head group, the length of the fatty acid chains, nor the hydrophile-lipophile balance (in the Triton X series) are major determinants for activation. Short-chain lecithins activate as monomers, whereas long-chain lecithins activate only above the critical micelle concentrations, suggesting high-affinity hydrophobic binding sites. Judged by EGTA inhibition, the amphiphile-mediated activation requires Ca 2+ in the absence of calmodulin. In addition, amphiphiles sensitize the adenylate cyclase to Ca 2+/calmodulin and are also synergistic with calmodulin for maximal stimulation.

Enzyme-linked immunosorbent assay for detection of pertussis immunoglobulin A in nasopharyngeal secretions as an indicator of recent infection.

An enzyme-linked immunosorbent assay was developed for detection of immunoglobulin A (IgA) antibody to Bordetella pertussis (PsIgA) in nasopharyngeal secretions as an indicator of recent infection. Secretion specimens submitted for pertussis culture were examined for PsIgA by this technique. Of 348 specimens tested, B. pertussis was cultured from 57, and PsIgA was detected in 8 culture-positive and 40 culture-negative specimens. The average time between onset of symptoms and specimen collection for the culture-positive, PsIgA-negative specimens was 10 days; for the culture-positive, PsIgA-positive specimens, 15 days; and for the culture-negative, PsIgA-positive specimens, 36 days. Examination of paired samples available from several culture-proven cases demonstrated conversion from a negative PsIgA in the early sample to a positive PsIgA in the follow-up sample. Our results indicate that PsIgA is produced during natural human infection and does not arise as a result of parenteral vaccination. PsIgA usually appears in the nasopharyngeal secretions during the second or third week of illness and persists for at least 3 months. The detection of PsIgA in secretions may be a valuable diagnostic aid in culture-negative patients with pertussis.

Adjuvant for IgE Antibody and Islet-Activating Protein in <i>Bordetella pertussis</i>

A new purified protein, with a molecular weight of 77,000, isolated from the supernatant of Bordetella pertussis culture fluid and called islet-activating protein (IAP), had adjuvant activity for IgE antibody in addition to leukocytosis-promoting, histamine-sensitizing and islet-activating activities. None of the three subunits (F1, F2 and F3) was biologically active by itself. All of the four activities were recovered in the complexes of subunits F1.3 and F2.3. These results suggest that the activities are brought about by a common factor inherent in B. pertussis.

Lymphocytosis Induced in Mice by Supernatant Fluids of Bordetella pertussis Cultures: A Histopathological Study

Abstract Intravenous injection into mice of phase I Bordetella pertussis culture supernatants produces a marked lymphocytosis. The evolution of lymphocytosis was correlated with histopathological alterations in the lymphoid organs. The early phase, 17 hr to day 3, was associated with massive depletion of both lymphocytes from the white pulp of the spleen and of cortical thymocytes. There was a corresponding profound decrease of splenic and thymic weights. The later phase was associated with a marked decrease in lymphocytes of lymph nodes while concomitantly the spleen and thymus appeared to be repopulating. Both thymic-dependent and thymic-independent lymphocyte populations were mobilized from spleen and lymph nodes. There was a striking diminution in the number of lymphocytes contained within the walls of postcapillary venules (PCVS) of all lymph nodes between 2 and 7 days. The data suggest that initially the lymphocytosis is due predominantly to release of splenic lymphocytes and to a lesser extent of thymocytes into the circulation. Subsequently, the lymphocytosis is sustained by the depopulation of the lymph nodes. Lymph node depopulation is maintained by the failure of circulating lymphocytes to emigrate from the blood through the PCVS.

The effect of Bordetella pertussis on lymphocyte cyclic AMP metabolism.

Bordetella pertussis culture fractions produce decreased metabolic responses to isoproterenol and epinephrine in mice and rats, suggesting the possibility of systemic beta adrenergic blockade. The present study was undertaken to elucidate the mechanism of the alteration in adrenergic responsiveness and to clarify its relationship to other biological effects of the organism. Lymphocytes were selected as a suitable tissue because of the marked alteration in lymphocyte distribution in pertussis-treated mice and rats, suggesting a change in the surface properties of these cells. Human peripheral blood lymphocytes, purified by nylon fiber chromatography, were studied. In short incubation experiments (20 min or less) B. pertussis did not alter the cyclic AMP response to isoproterenol, prostaglandin E (PGE(1)), or methacholine. However, when cells were preincubated with B. pertussis for 90 min at 37 degrees C, the responses to all three agents were markedly inhibited. Although these observations provide direct confirmation of the ability of B. pertussis to inhibit catecholamine responsiveness, the fact that PGE(1) and methacholine responses were also inhibited suggests that blockade at the level of the beta adrenergic receptor is doubtful. The inhibitory activity was localized in a nondialyzable, protein-rich fraction that is precipitated from B. pertussis culture fluid by ammonium sulfate at 90% of saturation. The bulk of the activity was obtained in the load volume after 50,000 g centrifugation in a cesium chloride gradient, density 1.2-1.5 (fraction 4). Fraction 4 produced a change in lymphocyte hormonal responsiveness at concentrations as low as 5 ng/ml. The relationship between cyclic AMP inhibitory activity in isolated human cells and leukocytosis-producing activity in intact mice was studied. The two activities seemed to parallel one another quite closely until the final Sephadex G-150 fractionation step, in which the two activities were obtained in the same column fraction, but a greater recovery of the leukocytosis-producing activity was obtained. Additional purification will be required to establish conclusively whether the same macromolecule is responsible for both activities. The availability of a bacterial product that markedly inhibits cyclic AMP accumulation in purified lymphocytes may help to clarify the role of cyclic AMP in lymphocyte activation by antigen and nonspecific mitogens.

DISTRIBUTION OF LABELED LYMPH NODE CELLS IN MICE DURING THE LYMPHOCYTOSIS INDUCED BY BORDETELLA PERTUSSIS

The mechanism by which Bordetella pertussis organisms and their products induce lymphocytosis in mice was analyzed in terms of the localization of syngeneic Cr-51-labeled lymph node cells. Labeled lymphoid cells incubated in vitro with the supernatant of B. pertussis cultures and then injected intravenously into normal recipients, or labeled cells injected into pertussis-treated recipients were unable to "home" to lymphoid organs but persisted for long periods in the blood. In animals "equipped" with a population of Cr-51-labeled lymphoid cells, administration of B. pertussis organisms or culture supernatant effected a shift of radioactivity from lymph nodes and spleen into the peripheral blood, coincident with the lymphocytosis. In in vitro experiments it was found that the active principle could bind to both erythrocytes and lymphocytes and could spontaneously elute from these cells onto labeled lymphocytes which were then unable to home efficiently. The data suggest that Bordetella pertussis-induced lymphocytosis involves a reversible attachment of the pertussis factor onto the surfaces of lymphocytes which prevents their recirculation to lymphoid organs. Recirculating lymphocytes are presumably affected as they emerge from lymphoid organs to enter the blood.

Studies on Lymphocytes. XVIII. Mechanism of Lymphocytosis Induced by Supernatant Fluids of Bordetella pertussis Cultures

Abstract Supernatant fluid from liquid medium cultures of phase I Bordetella pertussis organisms (pertussis supernatant) when given to calves and sheep by intravenous injection produces a marked lymphocytosis. Thoracic duct and splenic vein cannulation was performed in some animals to study the mechanism of production of this lymphocytosis. There was a transient increased mobilization of lymphocytes from the lymphoid organs as seen by the increase in output of lymphocytes from thoracic duct and splenic vein within a few minutes following administration of pertussis supernatant. This was followed by a decrease in the thoracic duct cell output as long as the blood lymphocyte count remained elevated, indicating an inhibition of recirculation of lymphocytes from blood to lymph. There was no evidence of increased new cell production.

Studies on the ultrastructure of Bordetella pertussis. I. Morphology, origin, and biological activity of structures present in the extracellular fluid of liquid cultures of Bordetella pertussis.

Two distinct particles have been recognized in the extracellular fluid of B. pertussis cultures. Both appeared to arise from the surface (cell wall) of the organism. One of these, a membranous particle, seemed to derive from long projections on the organism composed of the outer membrane of the cell wall. The second particle, a fine filament, was not readily comparable with any previously described bacterial structure. The two particles could be separated from each other by gradient centrifugation in CsCl. Lymphocytosis-promoting factor and histamine-sensitizing activity were only associated with fractions containing the fine filaments.

Attempts at Isolation of Lymphocytosis-Producing Factor from Supernatant Fluids of Bordetella pertussis Cultures

SummaryThe B. pertussis culture supernatant fluid possessing lymphocytosis-producing factor, histamine-sensitizing factor, lethal and hypoglycemia-producing factors was subjected to Sephadex G-10 and Bio Gel P-100 column chromatography. When chromatographic fractionation was carried out at 4° some fractions, found to retain the lym-phocytosis-producing property, had lost the lethal and hypoglycemia producing factors, and had partially lost the histaminesensitizing factor. Observations indicate that lymphocytosis-producing factor is distinct from other toxic factors.We wish to acknowledge the technical assistance of Mr. Gerd Borner.

Method for chromosome preparation from mouse peripheral lymphocytes.

SummaryA method for chromosome preparations from mouse peripheral lymphocytes is described. This method is based on the mobilization of lymphocytes from lymphoid tissues to peripheral blood by pertussis vaccine or by supernatant fluid from B. pertussis culture, on the effective separation of leukocytes from the erythrocytes by sedimentation with Plasmagel® and on the use of phytohemagglutinin (PHA) as mitogenic agent. Large numbers of well-spread metaphase plates suitable for detailed chromosome analysis can be obtained from a single mouse without sacrificing the animal. The procedure can be applied repeatedly and it was successful with all strains of mice tested.

Attempts at Isolation of Lymphocytosis-Producing Factor from Supernatant Fluids of Bordetella pertussis Cultures

The B. pertussis culture supernatant fluid possessing lymphocytosisproducing factor, histamine-sensitizing factor, lethal and hypoglycemia-producing factors was subjected to Sephadex G-10 and Bio Gel P-100 column chromatography. When chromatographic fractionation was carried out at 4° some fractions, found to retain the lymphocytosis-producing property, had lost the lethal and hypoglycemia producing factors, and had partially lost the histaminesensitizing factor. Observations indicate that lymphocytosis-producing factor is distinct from other toxic factors.

Bordetella pertussis culture having only species factor 1.

Eldering, Grace (Michigan Department of Public Health, Grand Rapids), Jack Holwerda, and Julia Baker. Bordetella pertussis culture having only species factor 1. J. Bacteriol. 91:1759-1762. 1966.-Preston's description of Bordetella pertussis 353-Z as a culture possessing only factor 1 was confirmed by agglutination and agglutinin-adsorption tests. The ld(50) values of this culture for mice inoculated by the intracerebral route were approximately the same as that of B. pertussis 18-323, the challenge culture in mouse potency tests of pertussis vaccine. In mouse protection tests, vaccine prepared with the factor 1 culture protected as well against challenge with B. pertussis 18-323, which has a broad antigenic pattern, as did vaccines made with cultures possessing factors 2, 3, 4, and 5 in addition to factor 1. When two groups of similarly immunized mice were challenged with 353-Z and 18-323, respectively, much lower ed(50) values were obtained with the animals challenged with 353-Z. This was true whether a factor 1 vaccine or a vaccine with factors 1, 2, 3, 4, and 5 was tested.

Studies on the Hemagglutinin of Hemophilus Pertussis

Summary and Conclusions Hemagglutinin activity can be demonstrated in the early phases of cultivation of Hemophilus pertussis but disappears as the cultures approach maximal density. Previous work has shown that cells from cultures that have reached maximal density have high immunizing potency. Absorption of supernatants from sonically disrupted H. pertussis cell concentrates with chicken red blood cells removes all of the hemagglutinating activity without diminution of toxicity or agglutinogen titer. Supernatants, after complete removal of hemagglutinating activity by absorption on chicken cells, still retain a considerable portion of their protective antigenicity. A spontaneous precipitate that develops in supernatants contains hemagglutinating activity but does not actively immunize mice. Certain properties of the hemagglutinating principle have been described, and the suggestion is made that there may be at least two hemagglutinins. It is concluded that the hemagglutinating substance of H. pertussis cultures is not identical with the toxin, the agglutinogen, or the immunizing antigen.

Competitive elution of pertussis hemagglutinin.

SummaryThe hemagglutinating factor of Hemophilus pertussis culture supernatants can apparently be eluted from human erythrocytes by suspending such erythrocytes in a distilled-water extract of frozen and thawed red cells. Following the removal of the H. pertussis factor from agglutinated cells, these cells can be re-agglutinated by addition of fresh H. pertussis culture supernatant. The factor cannot be eluted by incubation of agglutinated cells at various temperatures, or by suspending the cells in salt-free isotonic glucose solution.

Comparison of Pertussis Cultures by Mouse Protection and Virulence Tests

THE successful use of intracerebral infection in mouse protection tests of pertussis vaccine 1, 2 has called attention to a new method for measuring the virulence and protective properties of single cultures of Hemophilus pertussis. The practical value of such a study is apparent in relation to the criteria for selection of cultures for vaccine preparation. In the present study, the object has been to rank a group of cultures according to virulence and according to mouse protective properties, and to determine whether any correlation could be found between the two characteristics.

INTRACUTANEOUS REACTIONS IN PERTUSSIS

The reports of Modigliani and de Villa 1 and of Orgel 2 in the diagnosis of pertussis by intracutaneous reactions encouraged the medical world to hope that progress was being made in the control of this disease. Modigliani and de Villa made intracutaneous injections of a suspension containing a loopful of pertussis culture in 1 c.c. of distilled water. No reaction was obtained in diseases other than whooping cough, while an inflammatory reaction formed constantly in thirty-eight children with pertussis. Normal children gave negative reactions. Three children who had been exposed to the disease, but as yet showed no symptoms, gave a positive intracutaneous reaction, and a few days later developed whooping cough. Orgel used a suspension of pertussis bacilli in salt solution, 2,000 million organisms per cubic centimeter. Among twenty-five children in various stages of pertussis, all but one reacted positively, this one being in the incubation period. Among